BLI-Research-SynBio-2016-session-2 - Examples of Design Projects

Example 1

Design: Air-freshening Bacteria

The main component of the air freshener Febreze is a compound called hydroxypropyl beta cyclodextrin. Cyclodextrin is an 8-sugar ringed molecule that is created as the result of an enzymatic conversion of starch. The water released when the Febreze is sprayed partially dissolves the odor. The odor is then able to form a complex in the hole in the center of the cyclodextrin. This new complex can not bind to the smell-receptors in the nose, so the odor becomes undetectable.

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Hydroxypropyl beta cyclodextrin (HP Beta CD) is the active ingredient in Febreze.

However, the problem with Febreze is that each individual spray can only trap a limited number of the odor molecules. Because of this, Febreze is often over-sprayed and wasted, because people would rather spray air freshener with abandon than wait for the cyclodextrin to do its job. Not only does this mean that people are wasting Febreze and their money in vain, but this much air freshener being sprayed into the air without any odor to latch onto is bad for the environment.

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There are three types of cyclodextrins, as shown above. The smallest has only 6 glucose units, and the largest -- the one used in Febreze -- has 8 glucose units. There are different enzymes, called CGTases, that degrade starch in order to produce the different cyclodextrins. To regulate which cyclodextrin is made when a CGTase degrades starch, scientists specified the different enzymes that create different cyclodextrin. The one used in Febreze is y-CGTase.

This design project would allow bacteria to create y-CGTase, the enzyme that is needed to be present to degrade starch in order to produce cyclodextrin. However, the y-CGTase would only be produced in the presence of benzylamine. This would mean that even if the air freshener was sprayed randomly, no cyclodextrin would be produced and no chemicals would be released unless an actual odor was present. Also, rather than spraying several times, a user could spray the air freshener once, and as long as benzylamine was present in the air, the bacteria would keep producing y-CGTase that would keep degrading the starch until all the benzylamine was gone. This saves both money and the environment.

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As shown in the diagram, the benzylamine activates the promoter for cgtA, which in turn activates the cgtA gene. This results in the production of y-CGTase, which degrades starch to produce cyclodextrin.

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The table indicates that when benzylamine is present,
y-CGTase will also be present, and when benzylamine
is not present CGTase will not be produced.

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The above is a two-bottle system, with one bottle containing starch and the other containing the engineered bacteria. When the nozzle is pressed, the spray released is a mixture of the starch and the bacteria.

However, there are potential problems with the device. For instance, if there are copious amounts of benzylamine in the environment, the bacteria will keep creating the CGTase enzyme. However, the starch may run out before all the benzylamine is trapped. In this case there will be CGTase in the environment as well as the benzylamine. Testing the effectiveness of the system would be relatively simple: if the device was sprayed in the presence of benzylamine and the benzylamine was gone in a rational amount of time, it could be reasonably assumed that the device worked as it was intended to.

Example 2

Design Project: Insulin-generating Enteric Bacteria (IGEBs)

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Fig. 3 Process of blood glucose regulation in the human body

All persons affected by type 1 diabetes mellitus must receive injections of insulin or wear an insulin pump in order to survive, as their bodies donot produce insulin due to the destruction of beta cells in the islets of Langerhans.About 40% of those affected by type 2 diabetes (caused by insulin resistance) are treated with insulin injections. This treatment will focus mainly on type 1 diabetes, but could easily be used for the treatment of type 2 diabetes as well. The body regulates blood sugar primarily through a feedback cycle using insulin and glycogen (see Fig. 3). The release of insulin allows cells to take in sugar, lowering blood sugar. When blood sugar is too low, glycogen is released, prompting the liver to release sugars into the bloodstream. Type 1 diabetic patients do not produce insulin due to the destruction of beta-cells in the pancreas while type 2 diabetic patients are insulin resistant. The possibility of synthesizing bacteria that could exist within the body to produce insulin (or insulin substitutes) could be effectively used in order to treat diabetes, removing needle sticks, blood sugar highs and lows, and provide a more flexible and normal lifestyle for diabetics.
As previously stated, the current options for the treatment of type 1 diabetes mellitus are insulin injections and insulin pumps. Both provide problems and inconveniences for diabetic patients. Insulin injections require daily injections at various sites on the body, which are effective at delivering insulin but cause discomfort and be inconvenient for diabetics. Additionally, blood glucose highs and lows may occur as a result of insulin injections. However, they are more inexpensive and easier to use than insulin pumps. Insulin pumpsdeliver more precise amounts of insulin and can be adjusted to suit lifestyles and are thus more flexible. However, insulin pumps require extensive training and they must be attached to the body at all times, causing inconvenienceand a constant reminder of diabetes. As methods of treating diabetes, both insulin injections and pumps are effective but cause daily inconveniences to diabetics.

Fig. 4 Diagram of the glyoxylate cycle

This design project focuses mainly on the problem of having enteric bacteria (in this case, E. coli) produce insulin at the proper times, i.e. in response to the consumption of glucose and other carbohydrates by the diabetic patient. In this design, the absence of glucose will inhibit the production of insulin. In the absence of glucose, bacteria such as E. coli undergo the glyoxylate cycle to synthesize carbohydrates (see Fig. 4). One of the intermediates in this process is succinate. The presence of succinate would inhibit the expression of the luxS gene which produces the luxS enzyme. In turn, the luxS enzyme produces the AI-2 signaling molecule. At a certain critical concentration in a process similar to quorum sensing, the E. coliwould produce large amounts of insulin. AI-2 induces the Lsr transport cassette, which transports the AI-2 into the cell where it is phosphorylated. The phospho-A1-2 then binds to the LsrR repressor protein, releasing it from the lsr promoter, thereby allowing the open reading frame for the production of insulin to expressed. Thus, insulin will be produced when glucose is present (see Fig. 5). One of the benefits of this system is that bacteria will not continuously undergo the glyoxylate cycle in which case a small amount of insulin would be produced even when glucose is not present, mimicking the basal amount of insulin continuously produced by the pancreas or an insulin pump.

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Fig. 5 Proposed process for glucose-induced insulin production

For this system, if it were working “perfectly”, in the absence of glucose no insulin would be produced and in the presence of glucose insulin would be produced, as seen in the truth table below.

Glucose	Insulin production
0	0
1	1

However, in actuality, it is likely that a small amount of insulin would be produced even in the absence of glucose. In reality, however, this is not necessarily a negative. Insulin pumps, the technology on which this design is some what based produce a low level of basal insulin throughout the day to prevent highs and lows in blood glucose levels. Therefore, a small amount of insulin being produced continuously despite the absence of glucose (which is likely to occur in reality) is beneficial.

Glucose	Insulin production
absent	150 units
present	2000 units

IGEBs present many advantages over current technology in insulin therapy. The main disadvantages of insulin injections and insulin pumps are the inconveniences they provide for diabetics. Insulin pumps resolve many of the problems present with insulin injections, while providing disadvantages as well. IGEBs aim to resolve problems presented by insulin pumps. One of the main issues of insulin pumps is the physical pump that is constantly attached to the body, providing discomfort. IGEBs are a completely internally contained system, requiring no external devices. IGEBs also are completely self-adjusting, requiring no external output to adjust production of insulin levels. Insulin pumps require the patient to adjust insulin levels when eating or exercising. However, IGEBs will adjust without requiring human input, using the system described in the previous section. Additionally, IGEBs retain many of the advantages of insulin pumps, including a more flexible lifestyle and reducing blood sugar highs and lows.

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However, IGEBs also face many potential problems. One of the largest issues is how the bacteria will survive the gastrointestinal tract. If the bacteria are ingested (a similar idea to probiotics), the bacteria (or the capsule surrounding them) must be able to withstand the acidic conditions of the stomach. The next potential problem is the bacteria adhering to the villi in the small intestine without being flushed out by the body, which could require displacing the existing gut flora. Another issue is ensuring that enough insulin is absorbed into the bloodstream by the small intestine. Studies have shown that it is possible for insulin to be absorbed through the small intestine, but it is still unclear how much can be absorbed. Another issue is preventing the bacteria from mutating into a less than desirable form that could harm the patient, or transferring its genes through conjugation to other gut flora, which could result in the patient receiving too much insulin.

The testing of this system would involve observing the production of insulin by these bacteria in the absence and presence of glucose. The bacterial cells would be exposed to cycles of absence and presence of glucose of varying lengths of time and amounts of glucose. Insulin production would be tracked through these cycles to determine how much insulin is being produced and when. This testing would help adjust the bacterial insulin production to ideal levels for human insulin therapy. Further testing would also allow the development of technology to allow IGEBs to survive in the small intestine, providing an effective means for the treatment of diabetes mellitus.

Example 3

Design Project: N-Acyl Homoserine Lactone (N-AHL) Directed Bdellovibro bacteriovorus

The ProblemStewart’s wilt, also known as corn blight or bacterial blight, is caused by Pantoea stewartii. This plant pathogen is a facultative anaerobic, nonflagellate, nonspore-forming, nonmotile rod-shaped Gram-negative bacteria that seriously affects several corn types, such as sweet, dent, flint, flower, and popcorn. It is epidemic in the mid-Atlantic and Ohio River Valley areas and in the southern half of the Corn Belt. Other occurrences of the blight coincide with occurrence of the corn flea beetle, Chaetocnema pulicaria. The corn flea beetle is the primary vector of P. stewartii, which annually overwinters in the gut of the beetle. Each spring, adult beetles feast on fresh corn seedlings and transmit the blight via fecal contamination of the wounds they create on the plant. Surviving kernels from these infected specimens can also harbor the disease, resulting in a systemic infection of the seedling upon planting. Ultimately, Stewart’s’ wilt results in affected crops producing fewer and smaller ears of corn.

A corn flea beetle feasting on a leaf.

The greatest economic impacts of Stewart’s wilt on field corn occurred in the 1930’s, during a series of particularly hot summers and mild winters that resulted in thriving populations of flea corn beetles for several growing seasons, and during epidemics in the 1990’s when, Stewart’s wilt became a significant economic issue for the entire corn seed industry due to logistics of trading large volumes of field corn seed throughout the world. Today, economic impact upon field corn is minimal due to well-bred resistant hybrids. Most varieties of sweet corn, however, are still extremely susceptible to the blight and cause annual strain on sweet corn farmers in areas where the disease occurs. Yield losses in susceptible varieties of the corn after early-stage infections frequently range from 40 to 100%. Additional economic impacts of Stewart’s wilt include many phytosanitary regulations imposed upon sweet corn seed traders by trading partners. These regulations affect commerce by preventing seed export or adding additional costs to seed producers from areas known to harbor flea corn beetles.

Current Management Issues

The current prevention and management methods for Stewart’s wilt consist of spraying corn seeds, and then later the corn sprouts, with a cocktail of the insecticides clothianidin, imidacloprid and thiamethoxam. Repeated spraying is recommended in order to establish the presence of the insecticides within the field. The insecticides are not targeted at preventing or destroying P. stewartii, but aim to control the population of the corn flea beetle. Corn flea beetles are not an inherent threat to the health or growth of crops. The corn flea beetle injures corn plants by removing the outer surfaces of their leaves. These scratches rarely result in economic impact. Thetransmissionof P. stewartii to the corn is the primary motive behind controlling the corn flea beetle population. Thus, the main organism necessitating preventative action is P. stewartii, not the corn flea beetle.This has not been shown to decrease incidence or severity of the disease in susceptible varieties of corn.
All three of the aforementioned insecticides, however, belong to the class of neuro-active insecticides known as neonicotinoids. Neonicotinoids are chemically related to nicotine, and are the first new class of insecticides introduced in the last 50 years. Imidacloprid, in particular, is the most widely used insecticide in the world.

In March 2013, the American Bird Conservancy called for a ban on neonicotinoids and published a review of 200 studies showing their toxicity to birds, aquatic invertebrates, and other wildlife. Neonicotinoids have also been specifically named as the primary cause of honey-bee colony collapse disorder. In April 2013, the European Union voted to impose a two-year ban on neonicotinoid use in bee-attracting crops in hopes of seeing honey-bee population recovery. Although the Environmental Protection Agency has recognized the harmful and potentially long-term effects neonicotinoids have on honey-bee populations, no measures have been taken to restrict their use within the United States, partially do to the costs and efforts associated with using alternative forms of pest control.
So, Stewart's wilt is a problem, the corn flea beetles that transmit P. stewartii are not, and the current environmentally-toxic methods of controlling Stewart's wilt target the beetles, not the bacteria. Clearly there is room for improvement of the system. Suppose a technology could be developed in such a way that it targeted P. stewartii and prevented corn blight in an environmentally friendly way. It would need to be able to effectively locate P. stewartii within the wounds of corn leaves, then destroy it without the use of harsh chemicals that otherwise damaged the plant. In order to imagine such a technology, a greater understanding of how Stewart's wilt operates is required.

How the Pathogen WorksP. stewartii attacks the infected plant by colonizing the plant’s vascular system and producing extracellular polysaccharides (EPS) that cause obstruction of xylem vessels, which causes stunted growth, wilting leaves, withering, and death. The initial symptoms of Stewart’s wilt often appear as leaf legions surrounding flea beetle feeding scars. These pale legions then spread as streaks along leaf veins. These streaks eventually turn brown and necrotic, and yellow masses of bacteria can sometimes be seen oozing from the legions. Corn kernels become gray and dwarfed, and open cavities often form within the stalk, making the plant highly susceptible to stalk rot fungi in addition to Stewart’s wilt.
The EPS produced by P. stewartii are injected directly into corn cells via a type three protein secretion system (T3SS). Production of these EPS are regulated by quorum-sensing regulatory proteins that prevent the injection of EPS into the corn cells until a certain, effective population density of P. stewartii bacteria are present, ~2 X 10^8 cells/ml when grown in liquid culture.

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Although quorum-sensing systems in Gram-negative bacteria all reply on the same basic principle of interactions between N-acyl homoserine lactones (AHLs) and corresponding receptor proteins, the mechanisms and reactions that occur within each system vary, and to current knowledge each species of bacteria has an AHL with a slightly different shape due to differences in the composition of their R-group side-chain. Chain lengths vary from 4 to 18 carbon atoms and in the substitution of a carbonyl at the third carbon.
The quorum-sensing system in P. stewartii is primarily composed of the EsaI and EsaR proteins. EsaI is an N-acyl homoserine lactone (AHL) signal synthase, and EsaR is the cognate AHL-responsive transcription factor. The AHL produced by P. stewartii is N-(3-oxo-hexanoyl)-L-homoserine lactone (OHHL). When concentrations of OHHL in the extracellular matrix of P. stewartii are low, EsaR dimerizes and binds the DNA, thereby repressing target genes involved in synthesis of EPS. When concentrations of OHHL are great enough to be detected by the EsaR receptor proteins, EsaR binds in a 1:1 ratio with OHHL and detaches from the DNA sequence, allowing for the transcription of the rcsA gene, which codes proteins needed for EPS construction. In other words, the EsaR protein is a repressor, with derepression dependent upon OHHL. This fact will be important to the mechanics of the completed design.

The Solution: Another BacteriaBdellovibrio bacteriovorus is a predatory, obligate aerobic, Gram-negative bacteria discovered by Stolp and Petzhold in 1962. This comma-shaped bacteria is approximately 0.2 to 0.5μm wide and 0.5 to 2.5μm long and highly motile; specimens have been recorded swimming over 100 times their body length per second with the use of a single sheathed polar flagellum. B. bacteriovorus has been located in a wide range of terrestrial and aquatic environments such as soil, sewage, and river water.

The most distinct aspect of B. bacteriovorus is its biphasic predatory life-cycle. B. bacteriovorus preys solely on other Gram-negative bacteria in a parasitic manner. In the “attack phase,” B. bacteriovorus attaches to, and then lyses, the outer membrane of its prey. It then enters the cell, seals the membrane hole and forms a two-cell complex called a bdelloplast. B. bacteriovorus then breaks down the prey cell’s molecules, which it uses to elongate and form a filament. After exhausting available nutrients, the filament divides into several progeny Bdellovibrios. These progeny become mobile shortly before lysing the host cell and entering the open environment. They are then considered to be in the free living and motile phase, during which the traverse their environment in search of prey. The life cycle takes an average of one to three hours, and progeny cell number is dependent upon the size of prey in which they form.

The life cycle of B. bacteriovorus

Due to the necessity of prey for the reproduction of most strains of B. bacteriovorus, efficient motility is important to B. bacteriovorus. In Bdellovibrio bacteriovorus strain HD100, the wild-type strain on which this article will focus, genome sequencing and analysis have shown that there are three sets of motAB operons. These operons, Bd0144-Bd0145 (MotA1-MotB1), Bd3021-Bd3020 (MotA2-MotB2), and Bd3254-Bd3253 (MotA3-MotB3), code proteins involved in flagellar rotation.

MotAB protein pairs are transmembrane protein complexes which affect flagellar rotation by undergoing conformational changes in response to ion gradients maintained by the electron transport system of the cytoplasmic membrane. These changes act upon the rotor protein FliG, which causes rotation of the MS ring, rod, hook, and filament, thus causing the bacteria to swim.

Individual deletion of the motA gene sequences in a laboratory setting has revealed that each operon contributed to flagellar-mediated motility and that no single pair of proteins was essential for neither movement nor exit from the bdelloplast. The singular flagellum of B. bacteriovorus is thus thought to be powered by a hybrid motor controlled by all three pairs of stator proteins. However, deletion of MotA3 significantly reduced swimming speed by nearly a third compared to the minute speed differences observed in specimens with the deletion of MotA1 or MotA2. The MotAB3 operon therefore plays the most significant role in the functional ability of the bacterium’s flagella. Causality of the increased significance of the MotAB3 operon is believed to be related to aspartate resides in the MotB3 stator protein that are not found in MotB1 or MotB2.

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These MotAB protein complexes, however, only control the swimming power of B. bacteriovorus; they do not regulate swimming direction of the bacterium. B. bacteriovorus utilizes the “run and tumble” method of movement in which it alternates between phases of “running” or “smooth swimming” in a straight line and “tumbling” in a stationary rotation. This creates a pattern of movement known as a “random walk.” The change in movement occurs due to a change in flagellar rotation. A counter-clockwise rotation of the flagella of B. bacteriovorus causes a straight, smooth swim. A clockwise rotation induces tumbling.
Many bacteria, including B. bacteriovorus, have adapted a way of having a “biased random walk” that favors movement across a concentration gradient towards or away from particular chemicals so as to benefit or protect the bacteria. This phenomenon is known as chemotaxis.

The chemotactic system utilizes methyl-accepting chemotactic proteins (MCPs) which are transmembrane sensor proteins that detect molecules within the extracellular matrix. They react either directly with a ligand or with ligand-binding proteins, then transduce the signal down their hairpin structure to signaling proteins within the cytoplasm. These proteins, known as the Che complex, control the frequency the bacteria goes into the rotational “toggle” mode. Increasing concentrations of an attractant increase the likelihood that the bacteria will orient itself in a direction of positive chemotaxis, or towards the greatest level of concentration.

The Chemotaxis Protein System

The Synthetic ElementNow, hypothetically, if both the expression of the MotAB3 operon and the chemotactic system of B. bacteriovorus could be adapted to express and respond only to the particular N-Acyl homoserine lactone emitted by the EsaI proteins in P. stewartii, the statistical probability of B. bacteriovorus selecting P. stewartii for prey may increase to such an extent that the mutated B. bacteriovorus could be considered a unique biopesticide for that pathogen.

Two major alterations of the B. bacteriovorus genome would be necessary for such a specimen. First, the addition of the esaR gene such that the EsaR protein acts as a repressor of the MotAB3 operon, thereby making significant swimming speed dependent upon detection of OHHL molecules from P. stewartii. As previously discussed, the EsaR protein in P. stewartii acts as a repressor. Therefore when this protein is placed on the promoter of the MotAB operon, expression of the proteins MotA3 and MotB3 will not occur until the corresponding ligand for EsaR is detected.

Second, replacement of the natural methyl-accepting chemotaxis proteins with artificial MCP proteins that recognize N-(3-oxo-hexanoyl)-L-homoserine lactone as their ligand, preferably at a lower concentration than is required for detection by P. stewartii. Evolutionary adaptations have led to a wide range of MCPs that accept an equally diverse range of molecules as ligands. Thus, the creation of an AHL-accepting MCP with relative ease is not unrealistic. Once the AHL binds with this artificial MCP, it will set off the usual chain reaction of proteins within the chemotactic system that induce a biased walk towards the greatest concentration of AHLs, hence causing B. bacteriovorus to swim towards P. stewartii.

Because these two alterations of B. bacteriovorus are regulated by the same external chemical, the two responses will occur simultaneously when the AHLs of P. stewartii are present. Ideally, this concurrent swim speed increase and directional bias will “lock in” B. bacteriovorus to the desired prey. A simple truth table can be constructed:

Detection of OHHL by B. bacteriovorus	Positive Chemotaxis	Transcription of MotAB3	Mean Swim Speed (μm/s) +/- SD
0	0	0	63.2 +/- 5.5
1	1	1	26.5 +/- 1.8

ConsiderationsPotential problems with the design of the system arise when one considers that a preference and direction towards a specific prey does not translate to one-hundred percent certainty that the prey selected by B. bacteriovorus will be P. stewartii. Fortunately, testing of the engineered bacteria could be conducted with relative ease. Engineered B. bacteriovorus should be placed in an environment abundant in Gram-negative bacteria, including P. stewartii; predation levels on P. stewartii by the engineered B. bacteriovorus should then be compared to a control sample with an identical set-up in which the engineered bacteria were replaced with wild-type B. bacteriovorus HD100. Since neither B. bacteriovorus nor P. stewartii are human pathogens, only minimal safety precautions would be necessary for these trials.

Assuming optimal predatory behavior from the engineered B. bacteriovorus, this design, could eradicate the need for the use of heavy pesticides throughout the United States’ corn fields. Engineered B. bacteriovorus mixed into crop soil and/or sprayed on corn seedlings upon corn flea beetle sighting could potentially control the concentration of P. stewartii to such an extent that vascular colonization and thus crop devastation never occurs.

Since replication and multiplication of B. bacteriovorus occur each time a specimen successfully preys upon another bacterium, the populations of B. bacteriovorus should theoretically be sufficiently self-maintainable within a crop field and therefore not require reapplication within one growing season. However, since motility of the engineered bacterium will be compromised in the absence of P. stewartii, significant population survival beyond the point of management of Stewart’s wilt is unlikely.

Perhaps more importantly than management of Stewart’s wilt, this model for the adaptation of B. bacteriovorus could potentially be adapted to operate in response to any Gram-negative bacteria that uses an AHL-based quorum-sensing system, and may provide an alternative option for controlling antibiotic resistant strains of bacteria such as Pseudomonas aeruginosa or Staphylococcus aureus.